1. Field of the Invention
The present invention relates generally to the field of medical diagnosis and specifically for monitoring the presence of neoplastic diseases at an early stage to allow early therapeutic intervention.
2. Background Art
Currently, early detection of breast cancer in humans, particularly in women, depends on self-examination and mammography. However, routine mammography is not recommended for women under 50. Therefore, breast cancers in younger women tend not to be found until more advanced with a correspondingly poorer prognosis. Screening methods are needed to identify early stages of the transition of normal epithelial cells towards carcinoma in situ before the subsequent development of invasive and metastatic cancer.
Breast cancer appears to be genetically and/or morphologically, a heterogeneous disease and multiple mechanisms are responsible for the ultimate development of breast carcinoma from normal epithelial cells. The Her-2/neu (ERBB2/c-erbB-2) gene sequence (SEQ ID NO:9), hereinafter referred to as ERBB2, appears to be one of the primary genes responsible for the transition of normal epithelial cells towards carcinoma in situ and the subsequent development of invasive and metastatic cancer. However, by the time the gene product of ERBB2 is measurable, prognosis is not good. A means of identifying the initiation step for ERBB2 gene activity and interfering with that step are necessary for greater success in early identification and treatment of breast cancer.
Significant progress has been made at the molecular level to dissect the role of the ERBB2 gene and its association with breast cancer. However, mechanisms that control or initiate the activity of the ERBB2 gone have not been available to give early prediction or treatment of breast cancer. The results of some of these molecular studies are described herein.
Histologically, breast cancer comprises about 70-85% classified as ductal carcinema; the next largest subgroup is referred to as lobular carcinema. These two major classes of breast cancer comprise more than 80-95% of breast cancer in humans. It has been estimated that 5-15% of breast cancer in women under 50 years of age is associated with a genetic propensity for the disease..sup.1-13 Several recent studies have elucidated some of the inherited mechanisms which are at work in breast cancer..sup.14-17 A recent review has described various molecular determinates of growth, angiegenesis and metastases which may play a role in breast cancer..sup.18 In addition, the ERBB2 gone has recently been documented to be prognostically important in breast cancer..sup.43,45,56,69
The ERBB2 gone is the human counterpart of the rat neu oncogeno (SEQ ID NO:12), originally identified in ethyl nitreso-urea induced rat neurogiioblastomas by Weinberg and co-workers.sup.19,.degree. The ERBB2 oncegone codes for a protein of 185,000 dalton molecular weight (p185 product), and the product is similar in overall organization and primary amine acid sequence to the epidermal growth factor receptor (EGFR)..sup.21-23 A possible ligand for ERBB2 has recently been described..sup.24-26 The ERBB2 gone is not overexpressed in benign breast tissue,.sup.27 but significantly overexpressed in 60% of carcinema/in situ (proneoplastic lesion of breast carcinema) and in about 30% of invasive cancer..sup.28-30
The p185 product of the ERBB2 gone is a growth factor receptor with intrinsic protein tyrosine kinase activity.sup.31,32 which, when deregulated, or disregulated, results in unrestrained growth and cell transformation..sup.32-34 The transforming potential of the ERBB2 gone is also related to the levels of protein expression. This proto-oncogene is also frequently amplified in many human minors and in cell lines derived from tumors..sup.33,35-38 ERBB 2 gene overexpression in the absence of gene amplification has also been described..sup.33,36-38 The ERBB2 gene product is a potent oncoprotein when overexpressed in NIH-3T3 cells..sup.34 In a transgenic mouse model experiment, transgenic mice were created.sup.39,40 expressing the activated form of the rat neu proto-oncogene, under the control of steroid inducible promoter, and uniformly developed mammary adenocarcinoma. In addition, ERBB2 gene amplification in human breast tumor is often associated with poor patient prognosis..sup.33,38 The overexpression of ERBB2 has also been associated with poor prognosis in non-small cell lung cancer..sup.41,85
A convincing body of clinical and experimental evidence thus supports the role of ERBB2 protein in the progression of human cancers characterized by the overexpression of this oncogene product. Important aspects of this evidence include the poor prognosis of breast, ovarian and non-small cell carcinema patients whose tumors overexpress ERBB2 protein, as well as observations which indicate that modulation of ERBB2 protein activity by a momoclonal antibody can reverse many of the properties associated with tumor progression mediated by growth factor receptor..sup.42
A recent study.sup.43 of 209 consecutive female patients with invasive operable breast cancer from a defined urban population observed for a median of 30 years demonstrated that fifty-five patients (26%) had cancer and a positive ERBB2 oncoprotein stain reaction. They had significantly reduced 10 and 25 years survival rates as compared with those patients who had a negative stain reaction in their cancer (31% versus 48% and 31% versus 39% respectively with a P value=0.004). ERBB2 gene expression was also found to be associated with reduced survival among patients who had axillary nodal metastases (P value=0.003) but not among those patients who did not have metastases. ERBB2 expression was related to the ductal histologic type, poor histologic grade and high mitotic count, but not to tumor size, axillary nodal status, DNA ploidy or S-phase fraction. In a multivariate analysis among patients with nodal metastases, ERBB2 expression was found to be an independent prognostic factor (P value=0.004) that predicted poor survival. Based on these data, it was concluded that ERBB2 oncoprotein expression has long-term prognostic significance for predicting poor survival in breast cancer and it has an independent prognostic value among patients who presented with axillary nodal metastases. The mean survival time for the women with ERBB2 expressing group is only 29 months compared to the mean survival time of 110 months of the women with nonexpressing cancer. The difference between the survival curve is the greatest at approximately five years from the diagnosis (37% versus 64%) and diminished toward the end of the follow-up, which indicates that ERBB2 expressing cancers usually progress rapidly and are fatal. The result that ERBB2 expression predicts poor survival is contradictory to the opinion that it could only be a marker for drug resistance,.sup.44 not a marker for poor prognosis.
Overexpression of the ERBB2 oncogene has previously been correlated with poor prognosis in patients with infiltrating breast carcinema..sup.33 The authors reported a 35% difference in survival at four years for node positive patients with ERBB2 positive tumors..sup.33 This finding was emphasized in later studies with large numbers of patients..sup.45 It appears that the inconsistencies in the relationship between ERBB2 overexpression and mammary carcinoma are related to its correlation with tumor type. In studies of infiltrating carcinoma, the proportion of tumors showing overexpression has ranged from 10-30%;.sup.28-30,33,46-47 in carcinoma in situ, the incidence of overexpression is much higher, in the order of 60%..sup.28-30
Several studies.sup.44,48-50 have clearly shown that there is no loss of ERBB2 expression when invasive tumors progress from a pure in situ carcinoma. Therefore, there must be some other reason why fewer infiltrating tumors overexpress ERBB2. The nuclear sizes of the in situ and infiltrating components were also very similar and as has been found previously for in situ disease, almost all of the ERBB2 positive cases contained some large nuclei. A study.sup.51 has suggested that there are at least three groups of infiltrating tumors:
Group 1--those composed of cells with small nuclei which have arisen from small cell cribriform/micropapillary ductal carcinoma in situ. These have a low rate of proliferation and of ERBB2 overexpression.
Group 2--tumors composed of large cells which have arisen from large cell comedo ductal carcinoma in situ. These have a high rate of proliferation and ERBB2 overexpression.
Group 3--tumors composed of cells with variable nuclear sizes, but including some large nuclei, over half of which have a high rate of proliferation, but none of which overexpress ERBB2.
The hypothesis is that the latter group of tumors only have a transient in situ period and quickly become invasive. Because of this rapid progression to invasion, these tumors were not found in these studies of pure ductal carcinoma in situ. They made only a minor contribution to that study of tumors with a prominent ductal carcinoma in situ component accompanied by a variable infiltrating component but have become very obvious in this particular study. This could explain the dilution of overall ERBB2 positivity seen in studies of infiltrating tumors when compared to pure in situ tumors. If this is so, it could be accepted that the presence of ERBB2 overexpression is a marker of poor prognosis, since the ERBB2 positive in situ tumors are always composed of large cells, usually of comedo pattern and there are data to suggest that such tumors have a greater invasive potential than other patterns of in situ carcinoma..sup.52-55 In cases of infiltrating carcinoma, the ERBB2 positive tumors again contain large cells and are rapidly proliferating, both factors being associated with a poor prognosis. Whereas tumors with small nuclei and tumors with low proliferative activity are nearly always ERBB2 negative, there are also significant numbers of ERBB2 negative minors which contain at least some large cells, and many of these tumors have a high rate of proliferation. As already suggested, it is possible that this group of tumors has only a transient in situ stage.
Finally, another recent study.sup.56 demonstrated that tumors from 16% of the node negative patients and 19% of the node positive patients were ERBB2 positive. In both groups, ERBB2 positively correlated with negative progesterone receptor, negative estrogen receptors and high minor grade. The expression of ERBB2 was prognostically significant for node positive, but not for node negative patients. Tumors with overexpression of ERBB2 oncogene were less responsive to cyclophosphamide methotrexate and fluorouracil containing adjuvant therapy regimens than those with a normal mount of gene product, suggesting worse tumor behavior. For node positive patients, the effect of prolonged duration therapy on disease free survival was greater for patients without ERBB2 overexpression than those with ERBB2 overexpression. Similarly, for node negative patients, the effect of perioperative treatment on disease free survival was greater for those without ERBB2 overexpression than for those with ERBB2 overexpression.
U.S. Pat. Nos. 4,935,341 to Burgmann et al., issued Jun. 19, 1990, 4,968,603 to Simon et al. issued Nov. 6, 1990 and 5,183,884 to Kraus et al., issued Feb. 2, 1993, provide methods relating to the identification of ERBB2 gene expression, overexpression and prognostic indicators of breast cancer based on the ERBB2 gene product. The Slamon et al. '603 patent discloses amplification of the ERBB2 oncogene and its relationship to the status of breast and ovarian adenocarcinomas. In particular, the degree of gene amplification provides prognostic utility for breast cancer. The Bargrnann et al. '341 patent discloses mutations in the ERBB2 gene which result in an oncogenic state and provide an oligonucleotide probe capable of hybridizing to the mutated region. The Kraus et al. '884 patent discloses a DNA fragment distinct from EGFR and the ERBB2 gene, designated as ERBB-3. Marked elevation of ERBB-3 mRNA levels were demonstrated in certain human mammary tumor cell lines.
The above research and patents do not provide information that allows screening to identify earlier stages of the transition of normal epithelial cells towards carcinoma in situ before the subsequent development of invasive and metastatic cancer. These results indicate that the ERBB2 gene is extremely important in a significant percentage of breast cancers and the regulation of expression is perhaps a key determining factor in breast cancer development and progression. If the regulation can be controlled, transition to a cancerous state can be stopped.
Recent studies of cloning and characterization of an ERBB2 promoter have compared mouse neu promoter (SEQ ID NO:15) with human ERBB2 promoter..sup.57 (SEQ ID NO:10; SEQ ID NO:11) The presence of CAAT box and lack of a TATAA motif is one way in which the mouse neu promoter differs from the human ERBB2 promoter.sup.58 but is similar to the rat neu promoter..sup.59 (SEQ ID NO:13; SEQ ID NO:14) The GGA repeats observed between -204 and -184 (with respect to the translational start "ATG" codon) of the mouse neu promoter are also seen in rat.sup.59 neu and human ERBB2 promoters..sup.58 A sequence consensus for SP1 is located at -211 of the mouse neu promoter. SP1 consensus sequences are also seen in rat neu promoter and the human ERBB2 promoter in an analogous region. The sequence GCCGCCGC at -140 in the mouse neu promoter is similar to the binding site for G-CSF.sup.60 and is also observed in the rat neu promoter but not in the human ERBB2 promoter. A sequence similar to the OTF 1 motif,.sup.61,62 but differing by one nucleotide (ATGCAAAC instead of ATGCAAAT), is located at position -462. A similar sequence is also seen in the rat neu promoter and human ERBB2 promoters at equivalent positions. Sequences with homology to the AP2 consensus sequence (YSSCCMNSSS) (SEQ ID NO:16).sup.63 are located at -328 and -106 of the mouse neu promoter gene; similar sequences are also found in the corresponding regions of the rat neu promoter and human ERBB2 promoter.
A novel transcription factor termed "RNF".sup.64 was found to bind to the promoter of the rat neu gene. The binding sequence for this factor is also present in both the mouse (-439) neu promoter and human ERBB2 promoter. The GGTGGGGGGG sequence, (SEQ ID NO. 17) termed "GTG" enhancer, which is involved in autorepression of the rat neu transcription.sup.59 is located at position -249 to -240 in the mouse neu promoter. However, the corresponding region of the human ERBB2 promoter is different. Conservation of transcription factor sequences among these three species may imply a conserved function. It is not known at the present time whether those sequences that are different between rodent and human genes such as CAAT and TATAA box, GTG enhancer and other motifs might represent species specific functions.
This information, together with the fact that multiple transcriptional initiation sites arc mapped in both the rat neu and human ERBB2 genes, makes it likely that the TATAA sequence in the human ERBB2 promoter does not function as a transcriptional TATAA box. The previous studies on rat neu and human ERBB2 promoters focused mainly on a region within 1 Kb upstream from the transcriptional initiation sites. The current studies on the mouse neu promoter.sup.57 have lead to identification of a silencer region approximately three Kb upstream from the transcriptional initiation site, similar sequences have not yet been reported in human ERBB2 promoter. An estrogen responsive region has been found within the rat neu promoter region.sup.70
It has been reported that the expression of the ERBB2 gene is tissue specific and developmentally regulated..sup.65 Transcriptional regulation, therefore, may be one of the mechanisms (factor) leading to overexpression of ERBB2 gene in human cancer cells. Therefore, regardless of the relative distances from the transcriptional initiation site, identification of silencer and enhancer sequences controlling ERBB2 transcription provides important information that may allow clinical information to be obtained for studying transcriptional mechanisms resulting in cancer and understanding the biological role of ERBB2 gene regulation in breast cancer development, heterogeneity, progression and recurrence.
Primary gene induction or repression in eukaryotes does not require de novo protein synthesis, suggesting the involvement of post-translational modifications as well. In a recent review,.sup.67 it was summarized that many different types of stimuli that affect gene expression also led to the activation of protein kinases; it is likely that transcription factor function will be directly regulated by phosphorylation. Even though other types of post-translational modifications will undoubtedly be important in regulating transcription factor function, phosphorylation seems to be one of the most important functions which has been studied recently..sup.67,68
In summary, first, a transcription factor can be sequestered in the cytoplasm and rendered inactive through lack of access to the target sequences. Phosphorylation of the factor itself, or a cytoplasmic anchor protein allows translocation of the transcription factor into the nucleus, where it acts, generally by binding to the DNA at a specific site by protein-DNA interaction..sup.73 Second, the DNA-binding activity of nuclear transcription factor can be modulated by phosphorylation either positively or negatively..sup.67-68 Third, phosphorylation can affect the interaction of transcription factor transactivation domains with the transcriptional machinery..sup.67-68 These possibilities are by no means mutually exclusive and in principle phosphorylation at multiple sites by different protein kinases can result in regulation at several distinct levels. Nuclear translocation of various transcription factors modulated by phosphorylation has been demonstrated recently..sup.72
It has been shown that in unstimulated cells, with the notable exception of B cells, NFxB (nuclear factor xB) is retained in the cytoplasm in an inactive complex with the intermediary protein (IxB), which cannot bind DNA..sup.73,74 In response to various stimuli, including the phorbol-ester TPA, the IxB-NFxB complex dissociates and NFxB DNA-binding activity is detected in the nucleus..sup.73 DNA binding activity can be revealed in unstimulated cytoplasmic extracts by a number of means including treatment with sodium deoxycholate, which dissociates the IxB-NFxB complex..sup.74 Therefore, there is much evidence to suggest that a transcription factor can be found in the cytoplasmic extracts, as well as in the nuclear extract..sup..delta. A phosphorylation-dephosphorylation mechanism for the translocation of transcription factor in numerous systems by protein kinase A and protein kinase C has been demonstrated as indicated earlier..sup.67-68 Almost every eukaryofic transcription factor that has been analyzed in detail has proved to be phosphorylated. In most cases, however, the functional consequences of such phosphorylations, if any, are largely unknown.
There are only a few possible mechanisms proposed for the regulation of ERBB2 gene expression which are summarized as follows:
(i) A recent report has suggested that the E3 region of adenovirus induces down regulation of epidermal growth factor receptor. A similar repression of ERBB2 expression has also been documented, however, the repressed expression of ERBB2 is not through the E3 region of the adenovirus. The repression of ERBB2 expression is accomplished by E1A gene product, and it specifically repressed ERBB2 gene expression at the RNA level.sup.75 and full basal promoter activity of ERBB2 gene has been shown to be retained by two fragments of the ERBB2 5' region (-759 to -724 and -396 to -24 base pair). PA1 (ii) Functional inactivation of both alleles of the retinoblastoma susceptibility gene (RB) plays an important role in the etiology of both sporadic and familial retinoblastomas and several other types of human cancers, including breast cancer..sup.76,77 The RB gene may have cell cycle control function..sup.78,79 RB protein function may vary during the cell cycle because it shows cell cycle dependent changes in phosphorylation and RB protein can be phosphorylated by the cell cycle kinase p34 cdc2..sup.80 RB protein can also complex with the transcription factor E2F and inhibit E2F binding to the promoters of several cellular proliferation related genes..sup.81 Recent studies revealed that RB protein can negatively regulate the immediate early genes of c-fos and c-myc expression at the transcriptional level in NIH-3T3 cells..sup.82,83 RB also stimulates the growth inhibitory factor TGF-.beta.1 expression in certain cell types and subsequently suppresses cell growth..sup.84 Taken together, all of these results suggest that RB may limit the progression of cells through the cell cycle by sequestering a variety of nuclear proteins involved in growth regulatory gene transcription. As indicated earlier the amplification and overexpression of ERBB2 is involved in human breast and lung cancers..sup.38,85 Interestingly, inactivation of the RB gene has also been implicated in the oncegenesis of human breast and lung cancers.sup.77,86 and may suggest the possible molecular link between RB and the ERBB2 gene in the development and progression of breast cancer. A recent study has shown that the RB protein can bind specifically with a GTG-GGGGGGG sequence (SEQ ID NO: 18) in the ERBB2 promoter and suppress the promoter function. TMs study has concluded that the RB protein suppresses ERBB2 induced transformation by suppressing the ERBB2 promoter activity..sup.87
(iii) An interesting feature of the human ERBB2 gene promoter is the presence of two different types of regulatory elements: a CAAT box and SP1 binding sites. Transcription from the three most downstream RNA start sites appears to be controlled by the CAAT box and the TATA box, because these are respectively about 30 bp and 80 bp upstream of the early start sites and these distances are consistent with those in many other eukaryotic promoters..sup.88 On the other hand, transcription from the fourth RNA start site located further upstream seems to be controlled at least partly by SP1. In contrast With the ERBB2 gene promoter, the promoter region of the human epidermal growth factor receptor (EGFR) gene does not contain either a TATA box or a CAAT box but has 5 SP1 binding sites. Therefore, the expression of the ERBB2 gene may be regulated by the transcription factor SP1, a CAAT box binding protein and a TATA box binding protein,.sup.89-91 whereas the expression of the EGFR gene seems to be regulated by SP1 but not by the latter two proteins.
Since the ERBB2 gene appears to be important in breast cancer, treatment modalities have been reported in the literature employing strategies which target this gene. A recent report.sup.71 used a monoclonal antibody coupled to a toxin to target the extracellular domains of the ERBB2 receptor protein which are overexpressed on human breast and ovarian tumor cells in vitro. However, this is again late in the stage of the transition of normal epithelial cells to cancer. As described earlier, ERBB2 expressing cancers usually progress rapidly and are fatal. Treatment and diagnosis needs to be at an earlier stage, while the cells are still only showing hyperplasia.